The overall goals of the program are to identify the viral and cellular factors involved in the establishment, maintenance and reactivation of herpes simplex virus type 1 latency, and to determine the roles of these factors in the latency process. For this purpose, the following three projects are proposed: Project 1 (D. Knipe) will utilize in situ detection methods to define the nature of the block in immediate-early gene expression during the establishment of latency. Efforts will also be made to identify the type(s) of neurons able to support latency. Viral gene expression in ganglia latently infected with mutants defective in several DNA replication genes will be examined to assess the roles of individual DNA replication genes on gene expression during the establishment and reactivation of latency. The effect of the immune response on gene expression, virus replication and virus spread will be examined by histochemical techniques. Project 2 (D. Coen) will entail the development and use of sensitive PCR procedures to quantify different configurations of viral DNA in latently infected ganglia. Ganglia latently infected with defined mutants will be examined for levels and configurations of viral DNA and compared with viral gene expression by the same mutants (as determined in Projects 1 and 3). Quantitative RNA PCR and cDNA cloning will be used to characterize rare HSV transcripts during the establishment of latency by thymidine kinase (TK) mutants. Ganglia latently infected with mutants defective in TK and ribonucleotide reductase will be examined by in situ hybridization and PCR to determine at what level viral gene expression is blocked in reactivation of such mutants. The roles of individual functional properties of TK and viral DNA polymerase in replication, reactivation and pathogenesis in the peripheral and central nervous systems will be compared. Project 3 (P. Schaffer). Based on the observations that ICPO and the latency-associated transcripts (LATs) are able to stimulate HSV gene expression, and are also required for efficient reactivation from latency, the regions of these two genes that specify transactivating functions involved in reactivation in vivo will be identified and fine-mapped by standard genetic procedures. Efforts will also be made to clone and characterize the genes that specify the recently-identified cellular activating function(s) able to substitute for ICPO during productive infection and able to stimulate viral IE promoters. Collectively, the results of these studies will provide new information concerning the roles of viral and cellular regulatory proteins, and of viral DNA replication proteins in the establishment and reactivation of latency.